A study of short utrophin isoforms in mice deficient for full-length utrophin

Utrophin can functionally replace dystrophin in dystrophin-deficient muscle and may have a role in a therapeutic strategy for Duchenne muscular dystrophy. This has resulted in many investigations of the full-length muscle form of utrophin; however, the short utrophins and non-muscle forms have been relatively neglected, partly because they are difficult to analyze in the presence of the full-length form. Our study circumvents this problem by using mice deficient for the full-length form (UKOex6 mice) to study the translation and distribution of short utrophins. Four tissues were examined—kidney, testis, fetal hands/feet, and brain—and three novel short isoforms were identified, including Up120, which appears to be specific to kidney glomeruli, and Up 109, expressed in the fetal dermis. A third form, Up103, was found in testis but at extremely low levels. A cDNA for Up109 has been isolated and shown to have a unique NH2-terminal sequence. In addition, the first exons of Up109 and another short form, G-utrophin, have both been located within intron 55, 56 kb apart. Our immunological studies show that G-utrophin protein accumulates only in neural tissue, in line with its similarly restricted RNA distribution. Our study of testis expression shows, for the first time, that full-length utrophin is expressed at high levels in Leydig cells, raising the possibility that this protein is involved in testosterone secretion. We note that translation of the short utrophins, especially Up140 and Up71, is relatively inefficient and discuss the significance of this observation.     

Tenascin Mr 220,000 isoform expression correlates with corneal cell migration.

The three isoforms of chicken tenascin, an extracellular matrix glycoprotein, are generated by alternatively spliced fibronectin type III domains. The resulting proteins migrate as bands of Mr 220,000 (ten220), Mr 200,000 (ten200) and Mr 190,000 (ten190) on SDS-PAGE. We describe here two monoclonal antibodies, one specific for ten220 (mAb T17) and another that recognizes all isoforms (mAb T16). These were used to examine the differential expression of isoforms during development. Most impressive is the close correlation between ten220 expression and cell migration in the embryonic cornea. Initially (stage 18), ten190/200 can be detected within the corneal epithelium and along the basement membranes of the lens and sclera. Ten220 appears within the primary stroma immediately prior to the invasion by neural-crest-derived cells. This expression is maintained during the subsequent migration of fibroblasts from the conjunctiva into the primary stroma. With the completion of migration and the marked increase in matrix synthesis by corneal fibroblasts, ten220 disappears. Ten190/200 remains in the region adjoining the endothelium, the Bowman's membrane and the adjacent stroma. The cell-migration-associated isoform is isolated from extracts of embryonic tissues as a homohexamer. Low molecular weight forms appeared absent but a new tenascin band of Mr 210,000 could be detected in brain extracts which may be a new isoform. We conclude that the synthesis of tenascin isoforms is under tight developmental control and speculate that a function of the additional domains is to facilitate cell migration.     

Regional dysfunction correlates with myofiber disarray in transgenic mice with ventricular expression of ras

A hallmark of certain cardiac diseases such as familial hypertrophic cardiomyopathy is focal myofiber disarray. Regional ventricular dysfunction occurs in human subjects with hypertrophic cardiomyopathy; however, no direct evidence exists to correlate regional dysfunction with myofiber disarray. We used a transgenic mouse, which exhibits regional myofiber disarray via ventricular expression of the human oncogene ras, to investigate the relationship between myofiber disarray and septal surface strain. An isolated ejecting mouse heart preparation was used to record deformation of markers on the septal surface and to determine nonhomogeneous septal surface strain maps. Myofiber disarray made in histological tissue sections was correlated with gradients in surface systolic shortening. Significantly smaller maximum principal shortening was associated with disarray located near the right ventricle (RV) septal surface. There was also significantly smaller surface shear strain associated with disarray located either near the RV surface or at the midwall. Because surface shear is a local indicator of torsion, we conclude that myofiber disarray is associated with reduced septal torsion and reduced surface shortening.     

Targeting of a tropomyosin isoform to short microfilaments associated with the Golgi complex

A growing body of evidence suggests that the Golgi complex contains an actin-based filament system. We have previously reported that one or more isoforms from the tropomyosin gene Tm5NM (also known as gamma-Tm), but not from either the alpha- or beta-Tm genes, are associated with Golgi-derived vesicles (Heimann et al., (1999). J. Biol. Chem. 274, 10743-10750). We now show that Tm5NM-2 is sorted specifically to the Golgi complex, whereas Tm5NM-1, which differs by a single alternatively spliced internal exon, is incorporated into stress fibers. Tm5NM-2 is localized to the Golgi complex consistently throughout the G1 phase of the cell cycle and it associates with Golgi membranes in a brefeldin A-sensitive and cytochalasin D-resistant manner. An actin antibody, which preferentially reacts with the ends of microfilaments, newly reveals a population of short actin filaments associated with the Golgi complex and particularly with Golgi-derived vesicles. Tm5NM-2 is also found on these short microfilaments. We conclude that an alternative splice choice can restrict the sorting of a tropomyosin isoform to short actin filaments associated with Golgi-derived vesicles. Our evidence points to a role for these Golgi-associated microfilaments in vesicle budding at the level of the Golgi complex.     

Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice

Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx) mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv) mice express a near-full length dystrophin protein at ～5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.     

Iron-independent specific protein expression pattern in the liver of HFE-deficient mice

Hereditary hemochromatosis type I is an autosomal-recessive iron overload disease associated with a mutation in HFE gene. The most common mutation, C282Y, disrupts the disulfide bond necessary for the association of HFE with beta-2-microglobulin and abrogates cell surface HFE expression. HFE-deficient mice develop iron overload indicating a central role of the protein in the pathogenesis of hereditary hemochromatosis type I. However, despite significant effort, the role of the HFE protein in iron metabolism is still unknown. To shed a light on the molecular mechanism of HFE-related hemochromatosis we studied protein expression changes elicited by HFE-deficiency in the liver which is the organ critical for the regulation of iron metabolism. We undertook a proteomic study comparing protein expression in the liver of HFE deficient mice with control animals. We compared HFE-deficient animals with control animals with identical iron levels obtained by dietary treatment to identify changes specific to HFE deficiency rather than iron loading. We found 11 proteins that were differentially expressed in the HFE-deficient liver using two-dimensional electrophoresis and mass spectrometry identification. Of particular interest were urinary proteins 1, 2 and 6, glutathione-S-transferase P1, selenium binding protein 2, sarcosine dehydrogenase and thioredoxin-like protein 2. Our data suggest possible involvement of lipocalins, TNF-alpha signaling and PPAR alpha regulatory pathway in the pathogenesis of hereditary hemochromatosis and suggest future targeted research addressing the roles of the identified candidate genes in the molecular mechanism of hereditary hemochromatosis.     

Functional expression of the short isoform of the murine leptin receptor Ob-Rc (muB1.219) inXenopus laevis oocytes

Leptin, a hormone mainly secreted by the adipose tissue, acts on the hypothalamus to regulate food intake and thermogenesis. Six leptin receptor isoforms have been identified and localized in different tissues. While it is clear that leptin action in the brain occurs by binding to the long receptor isoform, several studies have shown that the short isoforms could be involved in the transcellular transport of the hormone from the blood to the brain. Based on these works, we decided to investigate whether the murine short leptin receptor isoform Ob-Rc (muB1.219) could transport leptin when expressed in Xenopus laevis oocytes. MuB1.219 cRNA was injected into the oocytes and functional studies were performed by incubating the oocytes in the presence of 2.5 nM [125I]-leptin, under different conditions. Results showed that leptin binding to the injected oocytes was four to eight-fold higher than the binding to the non-injected oocytes. This was blocked by 250 nM of non-radiolabeled leptin, suggesting that the binding was specific. Leptin internalization was observed from 30 min incubation onwards. Coexpression of the human Na+/glucose cotransporter and the leptin receptor showed that leptin increased sugar uptake into the oocytes. These results demonstrate that the short leptin receptor Ob-Rc is able to mediate binding and internalization of the hormone when expressed in oocytes and that it may perform intracellular signaling.     

Identification of a novel actin binding site within the Dp71 dystrophin isoform

The Dp71 dystrophin isoform has recently been shown to localize to actin filament bundles in early myogenesis. We have identified an actin binding motif within Dp71 that is not found in other dystrophin isoforms. Actin overlay assays and transfection of COS-7 cells with fusion proteins of wild type and mutated Flag epitope-tagged Dp71 demonstrate that this motif is necessary and sufficient to direct localization of Dp71 to actin stress fibers. Furthermore, this localization is independent of alternative splicing which alters the C-terminus of the protein. The identification of an actin binding site suggests Dp71 may function to anchor membrane receptors to the cytoskeleton.     

Developmental expression pattern of oncostatin M receptor beta in mice

Oncostatin M (OSM), which is predominantly expressed in bone marrow, is a member of the interleukin-6 family of cytokines, and appears to play important roles in hematopoiesis and the development of the liver. Recently, specific beta subunit of OSM receptor (OSMRbeta) was isolated from LO cells originated from aorta-gonad-mesonephros (AGM) region. In this study, we performed in situ hybridization to explore the expression pattern of OSMRbeta during murine embryogenesis, postnatal development, and in adult tissues. At 11.5 days postcoitum (dpc), the expression of OSMRbeta was first detected in aortic endothelial cells of the AGM region. At 14.5dpc, its gene expression was clearly observed in the primordia of some organs, including liver, thymus, choroid plexus, and limb, and persisted into postnatal mice. After birth, its gene expression became detectable in the other organs, such as lymph node, bone, heart, kidney, small intestine, nasal cavity, and lung.     

Defining the expression pattern of the LGI1 gene in BAC transgenic mice

The LGI1 gene has been implicated in the development of epilepsy and the invasion phenotype of glial cells. Controversy over the specific tissue expression pattern of this gene has stemmed from conflicting reports generated using immunohistochemistry and the polymerase chain reaction. LGI1 is one of a four-member family of secreted proteins with high homology and here we demonstrate, using GFP-tagged constructs from the four LGI1family members, that commonly used antibodies against LGI1 cross-react with different family members. With the uncertainty surrounding the use of commercially available antibodies to truly establish the expression pattern of LGI1, we generated transgenic mice carrying the LGI1-containing BAC, RP23-127G7, which had been modified to express the GFP reporter gene under the control of the endogenous regulatory elements required for LGI1 expression. Three founder mice were generated, and immunohistochemistry was used to determine the tissue-specific pattern of expression. In the brain, distinct regions of glial and neuronal cell expression were identified, as well as the choriod plexus, which is largely pia-derived. In addition, strong expression levels were identified in glandular regions of the prostate, individual tubules in the kidney, sympathetic ganglia in the kidney, sebaceous glands in the skin, the islets of Langerhans, the endometrium, and the ovary and testes. All other major organs analyzed were negative. The pattern of reporter gene expression was identical in three individual founder mice, arguing against a position effect altering expression profile due to the integration site of the BAC.     

Satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice

To determine whether muscle satellite cells and utrophin are correlated with the degree of damage in mdx skeletal muscles, we measured the area of the degenerative region as an indicator of myofiber degeneration in the masseter, gastrocnemius, soleus, and diaphragm muscles of mdx mice. Furthermore, we analyzed the expression levels of the paired box homeotic gene 7 (pax7), m-cadherin (the makers of muscle satellite cells), and utrophin mRNA. We also investigated the immunolocalization of m-cadherin and utrophin proteins in the muscles of normal C57BL/10J (B10) and mdx mice. The expression level of pax7 mRNA and the percentage of m-cadherin-positive cells among the total number of cell nuclei in the muscle tissues in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there was no significant correlation between muscle damage and expression level for pax7 mRNA (R = –0.140), nor was there a correlation between muscle damage and the percentage of satellite cells among the total number of cell nuclei (R = –0.411) in the mdx mice. The expression level of utrophin mRNA and the intensity of immunostaining for utrophin in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there also was not a significant correlation between muscle damage and expression level of utrophin mRNA (R = 0.231) in the mdx mice, although upregulated utrophin was incorporated into the sarcolemma. These results suggest that satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice. dystrophy; pax7; m-cadherin; dystrophin-related proteins     

The influence of gradual water acidification on the oxygen consumption pattern of fish

1. Carp (Cyprinus carpio) and tilapia (Oreochromis mossambicus) were kept in a flow-through respirometer for a week, while continuous oxygen consumption measurements were carried out, at a constant water O₂ concentration.2. Our measurements showed that tilapia decreased its standard metabolic rate (SMR), its average oxygen consumption, and its maximum metabolic rate in acid water (pH 4.0), whereas carp did not.3. It is proposed, based on our results, that carp and tilapia probably have different strategies to survive pH depression episodes: whereas tilapia avoids the additional stress of exercise, carp tries to escape the acid conditions.     

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer

Background

Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting.

Results

In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells.

Conclusion

A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.     

Crural diaphragm inhibition during esophageal distension correlates with contraction of the esophageal longitudinal muscle in cats

Esophageal distension causes simultaneous relaxation of the lower esophageal sphincter (LES) and crural diaphragm. The mechanism of crural diaphragm relaxation during esophageal distension is not well understood. We studied the motion of crural and costal diaphragm along with the motion of the distal esophagus during esophageal distension-induced relaxation of the LES and crural diaphragm. Wire electrodes were surgically implanted into the crural and costal diaphragm in five cats. In two additional cats, radiopaque markers were also sutured into the outer wall of the distal esophagus to monitor esophageal shortening. Under light anesthesia, animals were placed on an X-ray fluoroscope to monitor the motion of the diaphragm and the distal esophagus by tracking the radiopaque markers. Crural and costal diaphragm electromyograms (EMGs) were recorded along with the esophageal, LES, and gastric pressures. A 2-cm balloon placed 5 cm above the LES was used for esophageal distension. Effects of baclofen, a GABA(B) agonist, were also studied. Esophageal distension induced LES relaxation and selective inhibition of the crural diaphragm EMG. The crural diaphragm moved in a craniocaudal direction with expiration and inspiration, respectively. Esophageal distension-induced inhibition of the crural EMG was associated with sustained cranial motion of the crural diaphragm and esophagus. Baclofen blocked distension-induced LES relaxation and crural diaphragm EMG inhibition along with the cranial motion of the crural diaphragm and the distal esophagus. There is a close temporal correlation between esophageal distension-mediated LES relaxation and crural diaphragm inhibition with the sustained cranial motion of the crural diaphragm. Stretch caused by the longitudinal muscle contraction of the esophagus during distension of the esophagus may be important in causing LES relaxation and crural diaphragm inhibition.